Aging-related changes in cartilage extracellular matrix, cellularity and cell function that may predispose to issue failure and inadequate repair responses have been documented. The OA paradox is that despite the aging-related deficiencies in cartilage cell function, OA cartilage pathology is the consequence of cell activation which does not result in successful tissue repair but in destruction and abnormal modification of existing matrix and production of matrix components that are not part of normal articular cartilage, indicative of an abnormal cell differentiation. We propose the hypothesis that cartilage aging is associated with loss, abnormal differentiation and dysfunction that differentially affects cartilage cell subpopulations. Mature chondrocytes are reduced through apoptotic mechanisms and compromised in function. Adult stem cells are present in aging cartilage but chondrocytic differentiation is compromised by aging-associated changes in signaling mechanisms and the nflammatory milieu of OA. AIM 1: Chondrocyte aging: an analysis of cartilage zones and cell subpopulations. artilage cell subpopulations (differentiated chondrocytes and progenitor cells) in specific zones of normal aging and OA cartilage will be identified using cell surface and differentiation markers. Aging-associated changes in frequency will be correlated with OA histopathology, and markers of abnormal chondrocyte differentiation. In vitro analysis of cell functions will be performed by stimulation of cartilage explants and in situ measurement of activation and differentiation markers. AIM 2: Sox9 expression and activation. Sox9 is a key regulator of chondrocyte differentiation and cartilage specific gene expression but its role in articular chondrocyte differentiation and in activation of differentiated chondrocytes remains to be established. Preliminary studies suggest changes in Sox9 expression in aging and OA chondrocytes. The proposed studies will address details of Sox9 expression and activation in aging and OAchondrocytes. AIM 3: TGFG activation of Sox9: role in chondrocyte activation and differentiation and changes in aging. TGFG regulates chondrogenesis in mesenchymal stem cells and this is in part mediated via Smad3 dependent Sox9 activation and by MAP kinase pathways.TGFC. also activates differentiated articular chondrocytes but these responses are compromised with aging. This aim will address the role of Smad and MAP kinase signaling and Sox9 in TGFB-mediated activation of normal and aging chondrocytes. The results from the proposed studies will add important new information on chondrocyte biology, cartilage aging and osteoarthritis pathophysiology.